Two coloured fluorimetric protease assay

Abstract

The invention concerns an autofluorescent fusion protein suitable for use as a protease substrate, a nucleic acid sequence which encodes this fusion protein, and a method of using the fusion protein and/or the nucleic acid sequence in a dual color, confocal fluorometric assay for the detection and quantification of proteolytic activity or proteolytic inhibitory activity in samples or cells.

Description

[0001] The invention described herein concerns an autofluorescent fusion protein, which is suitable for use as a protease substrate, a nucleic acid sequence which encodes this fusion protein and a method using the fusion protein and / or the nucleic acid sequence in a dual-colour, confocal fluorometric assay for the detection and quantification of proteolytic activity in liquid samples or cells. BACKGROUND OF THE INVENTION [0002] Proteases are enzymes which catalyze the hydrolytic cleavage of peptide molecules. The detection and quantitative determination of proteolytic activity is of significance for various research fields as well as for the pharmaceutical and biotechnical industries. Applicable test methods, known as protease assays, are used in the search for new enzymes with proteolytic activity, for their biochemical characterization, for the contamination control in production equipment and in the search for substances with activity modifying properties. Particular focus here i...

AI Technical Summary

Benefits of technology

[0039]FIG. 3: Measurement of the two-photon cross-correlation during the cleavage of the fusion protein in variant 2 (fusion protein B; SEQ ID NO:2) as a consequence of the effect of the TEV protease. The cleavage of the bond and the consequent reduction of the proportion of double fluorescing molecules in the measurement solution cause the decrease of the cross-correlation amplitude.

Problems solved by technology

Besides the disadvantage of the chronological disparity between reaction and determination of the measurement values, these methods are characterized especially by the complex handling.

However, these substrates of the first generation suffer from a number of disadvantages which limit their use.

This drastically reduces the enzyme's selectivity and the transformation rate.

These substrates are completely unsuitable for the analysis of substrate specificities on the peptide chain at some distance from the protease cleavage site, i.e., the secondary substrate specificity.

But the cleavage reaction separates the quencher and the fluorophore, causing a strong rise in the measurable fluorescence emission resulting from the excitation of the fluorophore.

Consequently, these three parameters restrict the applicability of such protease substrates.

However, a disadvantage of such known protease substrates with chemically linked fluorophores is the complex method required to synthesize them.

Although peptides can be synthesized relatively efficiently by solid-phase synthesis up to a length of about 50 amino acids, the double, site-specific coupling of fluorophores requires considerable effort, as the fluorophores used are usually not compatible with the peptide synthesis chemistry.

Furthermore, none of the substrates known to date does meet the requirements of an intracellular protease assay.

Disruption of the cells with the goal of making the proteases accessible to measurement is usually a complex process.

In addition, the spatial resolution is lost.

These methods, however, all have the problem that they are relatively labor-intensive, that they principally damage the cells and that a potential control of quantity and site of the insertion is difficult.

Nevertheless, it is subject to all of the disadvantages of fluorescence energy transfer substrates described above, such as the restriction in the structural design of the substrate and the maximum possible length of the protease recognition sequence.

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